Adenosine receptor ligands have anti-inflammatory effects and modulate immune responses by up-regulating IL-10 production by immunostimulated macrophages. The adenosine receptor family comprises G protein-coupled heptahelical transmembrane receptors classified into four types: A 1, A 2A, A 2B, and A 3. Our understanding of the signaling mechanisms leading to enhanced IL-10 production following adenosine receptor occupancy on macrophages is limited. In this study, we demonstrate that adenosine receptor occupancy increases IL-10 production by LPS-stimulated macrophages without affecting IL-10 promoter activity and IL-10 mRNA levels, indicating a posttranscriptional mechanism. Transfection experiments with reporter constructs containing sequences corresponding to the AU-rich 3′-untranslated region (UTR) of IL-10 mRNA confirmed that adenosine receptor activation acts by relieving the translational repressive effect of the IL-10 3′-UTR. By contrast, adenosine receptor activation failed to liberate the translational arrest conferred by the 3′-UTR of TNF-α mRNA. The IL-10 3′-UTR formed specific complexes with proteins present in cytoplasmic extracts of RAW 264.7 cells. Adenosine enhanced binding of proteins to a region of the IL-10 3′-UTR containing the GUAUUUAUU nonamer. The stimulatory effect of adenosine on IL-10 production was mediated through the A 2B receptor, because the order of potency of selective agonists was 5′-N-ethylcarboxamidoadenosine (NECA)> N 6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA)> 2-chloro-N 6-cyclopentyladenosine (CCPA)= 2-p-(2-carboxyethyl) phenethylamino-5′-N-ethyl-carboxamidoadenosine (CGS-21680). Also, the selective A 2B antagonist, alloxazine, prevented the effect of adenosine. Collectively, these studies identify a novel pathway in which activation of a G protein-coupled receptor augments translation of an anti-inflammatory gene.